Acquired Immune Deficiency Syndrome (AIDS) caused by HIV infection is one of most significant health threat faced by the world. Currently, AIDS has almost spread to countries around the world, and resulted in more than 40 million patients suffering from it, and nearly 30 million people were killed by it (WHO, Report on the Global AIDS Epidemic, 2004). In recent years, the spread of AIDS in China is growing rapidly, and the infected people have already amounted to 0.84 million. At present, the treatment of HIV infection is primarily through high-intensity anti-retrovirus therapy, such as through a combined use of inhibitors against viral reverse transcriptase and protease. However, due to the high mutation rate of HIV and its complex pathogenesis, this type of approach could not completely eradicate the virus in vivo. Therefore, there is an urgent need to develop a new method of treatment for dealing with the threat of AIDS.
RNA interference (RNAi) is a mechanism for inhibiting gene expression intracellularly mediated by a double-stranded RNA (dsRNA), and was first proposed in a research on the inhibition of gene expression in nematode in 1998 (Fire A et al., Nature, 1998, 391:806-811). Further research discovered that the RNAi exists widely in higher mammals and almost all eukaryotic organisms, such as fungi, arabidopsis, hydra, planarian, trypanosoma, zebrafish etc., and is a widely existing and conservative mechanism for inhibiting gene expression, which may play a role in the regulation of gene expression, protection against virus infection and suppression of the activities of transposon and so on (Dylexhoom D M et al., nature molecular biology review, 2003, 4: 457-467). Working mechanism of RNAi has now almost been elucidated: the endogenous or exogenous dsRNA molecules are cleaved into small interfering RNA (siRNA) in the cytoplasm by Dicer belonging to RNase III. Typical siRNA structural characteristics are: a dsRNA that is 19-23 nt in length with its 5′ end phosphorylated and its 3′ end symmetrically overhanged by 2-3 nt and with hydroxyl. siRNA molecules bind to the protein complex of RNA-inducing silencing complex (RISC), and the RISC has the helicase and endonuclease activities. The siRNA molecule is unwound in the complex, and the antisense strand can match target mRNA according to the principle of base pairing, and guide the RISC binding to it to enzymatically digest the target mRNA at a position 10 nt from the 5′ end in the middle of the antisense strand binding region, thereby inhibiting the expression of the target gene. Currently, main methods for obtaining siRNA include: plasmid and recombinant virus vector that can express small hairpin RNA (shRNA), chemical synthesis, in vitro transcription and so on.
At present, RNAi has shown a good application prospect in the research of prevention and treatment of diseases such as viral disease, including AIDS, and tumor. Studies have shown that siRNA targeting the mRNA of HIV-1 can inhibit the replication of HIV-1 and viral gene expression in HIV-1 susceptible cell cultured in vitro (Martinez M A et al., Immunology Trends, 2002, 23: 559-561). Due to the complicated pathogenic mechanism of HIV, its effective treatment requires highly efficient inhibition of viral replication and gene expression. However, due to the different inhibition efficiency of different targets, not all RNA interference targets met the requirement of conventional design are able to inhibit the expression of target genes effectively. Therefore, suitable RNA interference targets having high inhibition efficiency becomes an important factor in successful HIV treatment using RNAi technology. Selection of an appropriate RNA interference target needs comprehensive considerations in structural features, inhibition efficiency, non-human gene homology and so on. Assistant methods available include the following methods that have been put forward presently: siRNA aided designing software, analysis of the molecular structure of RNA, nucleic acid sequence analysis and alignment and experimental experience etc., these also can be verified through particular inhibition experiment.
Development of novel and more effective AIDS treatment methods are expected based on RNA interference technology, and such kind of treatment method requires a RNA interference target that can effectively suppress HIV replication and expression to be provided. The present invention meets this need and provides a RNA interference target, a recombinant expression vector and so on for such purpose.